This project aims to determine physiologically critical functions of NF-kappaB proteins and their regulators in the context of specific biologic reactions. Regulators include the inhibitory IkappaB proteins, as well as proximal activators. Research is based on the discovery of specific defects (especially of the immune system) in mice rendered deficient for various NF- kappaB proteins or their regulators. The ultimate goal is to identify critical molecular targets of the NF-kappaB factors in specific immune responses and to identify the essential signals that activate the factors. Previously we have generated mice deficient in the NF-kappaB2-locus encoded proteins p52 and its precursor p100. In addition we have generated mice deficient in both NF-kappaB1 and NF-kappaB2, which are highly homologous. NF-kappaB2 knockout mice are impaired in secondary lymphoid architecture due to stromal defects but also exhibit some defects intrinsic to B cells. The NF-kappaB1 and NF-kappaB2 double knockout mutant mice are profoundly impaired in their immune system, with many defects not seen in either single knockout, consistent with redundant activities of these two proteins. Significantly, only the double knockout mice are completely blocked in the development of mature osteoclasts and of mature B cells. We further determined in adoptive transfer experiments that the blocks in generation of a mature B cells and mature osteoclasts are intrinsic to these cell lineages. We demonstrate now that NF-kappaB1 and NF-kappaB2 are not required to generate initial osteoclast progenitors in the bone, but that these progenitors, once formed, no longer respond correctly to stimulation with the TNF family member RANK ligand. RANK receptor stimulation is critical for the continued survival and differentiation of osteoclast progenitors into mature osteoclasts. We furthermore demonstrate now that B cell development is blocked during an early transitional stage of differentiation of B cells in spleen, shortly after entry from the bone marrow. This results in a complete absence of recirculating mature B cells. The double-deficient mutant B cells are intrinsically more apoptotic than their wild-type counterparts, indicating that NF-kappaB1,2 are essential to the survival of transitional splenic B cells. The contribution of NF-kappaB2 to survival could be shown to occur in response to the TNF-family member BAFF. BAFF induced the processing of the p100 precursor form of NF-kappaB2 to p52 and the processing was necessary for the extended survival of transitional B cells generated with in vitro bone marrow cultures. These results indicate that NF-kappaB2 processing, an alternative pathway of activating NF-kappaB, is involved in survival of transitional B cells in spleen, at a stage of development when only a few of these cells are selected into the mature pool of B cells and when self-tolerance is enforced.